Improved Electrospray Ionization Efﬁciency Compensates for Diminished Chromatographic Resolution and Enables Proteomics Analysis of Tyrosine Signaling in Embryonic Stem Cells Scott B. Ficarro, †,‡,§ Yi Zhang, †,‡ Yu Lu, †,§ Ahmadali R. Moghimi, † Manor Askenazi, †,‡,§,| Elzbieta Hyatt, ⊥ Eric D. Smith, †,‡ Leah Boyer, # Thorsten M. Schlaeger, # C. John Luckey, ⊥ and Jarrod A. Marto* ,†,‡,§ Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, 44 Binney Street, Smith 1158A, Boston, Massachusetts 02115-6084, Department of Biological Chemistry and Molecular Pharmacology, and Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, Stem Cell Program and Division of Hematology/Oncology, Children’s Hospital Boston, Boston, Massachusetts 02115, and Department of Biological Chemistry, The Hebrew University of Jerusalem, Israel Characterization of signaling pathways in embryonic stem cells is a prerequisite for future application of these cells to treat human disease and other disorders. Identiﬁcation of tyrosine signaling cascades is of particular interest but is complicated by the relatively low levels of tyrosine phosphorylation in embryonic stem cells. These hurdles correlate with the primary limitations of mass spectrometry- based proteomics; namely, poor detection limit and dynamic range. To overcome these obstacles, we fabri- cated miniaturized LC-electrospray assemblies that pro- vided ∼15-fold improvement in LC-MS performance. Signiﬁcantly, our characterization data demonstrate that electrospray ionization efﬁciency compensates for dimin- ished chromatographic performance at efﬂuent ﬂow rates below Van Deemter minima. Use of these assemblies facilitated quantitative proteomics-based analysis of ty- rosine signaling cascades in embryonic stem cells. Our results suggest that a renewed focus on miniaturized LC coupled to ultralow ﬂow electrospray will provide a viable path for proteomic analysis of primary cells and rare post- translational modiﬁcations. To date, pluripotent cells have been isolated from the inner cell mass of blastocysts (ESC), 1 cultured postimplantation epiblasts, 2,3 and reprogrammed differentiated ﬁbroblasts (iPS). 4-8 Each of these pluripotent cell populations can in principle undergo programmed differentiation to generate nearly any cell type. Thus for human pluripotent cells, there is growing excitement that these cells may form the basis of new therapies to ﬁght disease. The majority of large-scale data for ESC are based on genome- wide transcriptional proﬁles and interrogation of transcription factor binding sites. 9 By comparison a relatively small number of protein signaling pathways have been functionally linked to self- renewal and differentiation in ESC. For example, leukemia inhibitory factor (LIF) maintains self-renewal in murine ESC (mESC) via signaling through the receptor g130, resulting in STAT3 phosphorylation and activation, 10 while self-renewal in human ESC (hESC) is dependent upon basic ﬁbroblast growth factor (bFGF) signaling. 11,12 Similarly, activity of selected Src- family kinases, 13,14 glycogen synthethase kinase 3 beta (GSK), and phospho-inositol 3- kinase (PI3K) have been implicated in both self-renewal and differentiation. 15-17 Collectively, these results * To whom correspondence should be addressed. E-mail: jarrod_marto@ dfci.harvard.edu. Phone: (617) 632-3150. 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